Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

A first global analysis of plasmid encoded proteins in the ACLAME database

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.     

The evolution and suppression of male suicide under paternal genome elimination

Different genetic systems can be both the cause and the consequence of genetic conflict over the transmission of genes, obscuring their evolutionary origin. For instance, with paternal genome elimination (PGE), found in some insects and mites, both sexes develop from fertilized eggs, but in males the paternally derived chromosomes are either lost (embryonic PGE) or deactivated (germline PGE) during embryogenesis and not transmitted to the next generation. Evolution of germline PGE requires two transitions: (1) elimination of the paternal genome during spermatogenesis; (2) deactivation of the paternal genome early in development. Hypotheses for the evolution of PGE have mainly focused on the first transition. However, maternal genes seem to be responsible for the deactivation and here we investigate if maternal suppression could have evolved in response to paternally expressed male suicide genes. We show that sibling competition can cause such genes to spread quickly and that inbreeding is necessary to prevent fixation of male suicide, and subsequent population extinction. Once male-suicide has evolved, maternally expressed suppressor genes can invade in the population. Our results highlight the rich opportunity for genetic conflict in asymmetric genetic systems and the counterintuitive phenotypes that can evolve as a result.     

Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution

Restriction–modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.     

Symbiotic origin of a novel actin gene in the cryptophyte Pyrenomonas helgolandii

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment; PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids, with this genome ultimately being lost when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia)

Genome evolution of bacteria is usually influenced by ecology, such that bacteria with a free-living stage have large genomes and high rates of horizontal gene transfer, while obligate intracellular bacteria have small genomes with typically low amounts of gene exchange. However, recent studies indicate that obligate intracellular species that host-switch frequently harbor agents of horizontal transfer such as mobile elements. For example, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial coinfections in the same host. Here we show that despite the phage's rampant mobility between coinfections, the prophage's genome displays features of constraint related to its intracellular niche. First, there is always at least one intact prophage WO and usually several degenerate, independently-acquired WO prophages in each Wolbachia genome. Second, while the prophage genomes are modular in composition with genes of similar function grouping together, the modules are generally not interchangeable with other unrelated phages and thus do not evolve by the Modular Theory. Third, there is an unusual core genome that strictly consists of head and baseplate genes; other gene modules are frequently deleted. Fourth, the prophage recombinases are diverse and there is no conserved integration sequence. Finally, the molecular evolutionary forces acting on prophage WO are point mutation, intragenic recombination, deletion, and purifying selection. Taken together, these analyses indicate that while lateral transfer of phage WO is pervasive between Wolbachia with occasional new gene uptake, constraints of the intracellular niche obstruct extensive mixture between WO and the global phage population. Although the Modular Theory has long been considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant in phages of obligate intracellular bacteria.     

Neutral drift and polymorphism in gene-for-gene systems

Pathogens are a main driving force of the evolution of plants and animals. Being resistant to diseases confers a high selective advantage to hosts, yet many host–pathogen systems show a remarkable degree of polymorphism of host resistance and pathogen virulence. The most common explanation of this phenomenon is that both resistance and virulence genes are costly and that there is selection against those genes when they are unnecessary. Here, we use stochastic multi‐locus simulations to show that the origin and the maintenance of genetic polymorphism in plant–pathogen systems can be explained without costs. In multi‐locus gene‐for‐gene systems, temporal domination of a super pathogen can cause polymorphism in resistance through neutral drift. With an increasing number of susceptible alleles in the host population, pathogen types other than the super race are able to cause infections and invade the population, leading to higher pathogen diversity and in turn to higher host diversity.     

Amplicon Remodeling and Genomic Mutations Drive Population Dynamics after Segmental Amplification

New enzymes often evolve by duplication and divergence of genes encoding enzymes with promiscuous activities that have become important in the face of environmental opportunities or challenges. Amplifications that increase the copy number of the gene under selection commonly amplify many surrounding genes. Extra copies of these coamplified genes must be removed, either during or after evolution of a new enzyme. Here we report that amplicon remodeling can begin even before mutations occur in the gene under selection. Amplicon remodeling and mutations elsewhere in the genome that indirectly increase fitness result in complex population dynamics, leading to emergence of clones that have improved fitness by different mechanisms. In this work, one of the two most successful clones had undergone two episodes of amplicon remodeling, leaving only four coamplified genes surrounding the gene under selection. Amplicon remodeling in the other clone resulted in removal of 111 genes from the genome, an acceptable solution under these selection conditions, but one that would certainly impair fitness under other environmental conditions.     

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity

Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.     

Young intragenic miRNAs are less coexpressed with host genes than old ones: implications of miRNA-host gene coevolution

MicroRNAs (miRNAs) have emerged as key regulators of gene expression. Intragenic miRNAs account for ～50% of mammalian miRNAs. Classic studies reported that they are usually coexpressed with host genes. Here, using genome-wide miRNA and gene expression profiles from five sample sets, we show that evolutionarily conserved ('old') intragenic miRNAs tend to be coexpressed with host genes, but non-conserved ('young') ones rarely do so. This result is robust: in all sample sets, the coexpression rate of young miRNAs is significantly lower than that of conserved ones even after controlling for abundance. As a result, although young miRNAs dominate in human genome, the majority of intragenic miRNAs that show coexpression with host genes are phylogenetically old ones. For younger miRNAs, extrapolation of their expression profiles from those of their host genes should be treated with caution. We propose a model to explain this phenomenon in which the majority of young miRNAs are unlikely to be coexpressed with host genes; however, for some fraction of young miRNAs coexpression with their host genes, initially imbued by chromatin level effects, is advantageous and these are the ones likely to embed into the system and evolve ever higher levels of coexpression, possibly by evolving piggybacking mechanisms.     

The heritage of pathogen pressures and ancient demography in the human innate-immunity CD209/CD209L region

The innate immunity system constitutes the first line of host defense against pathogens. Two closely related innate immunity genes, CD209 and CD209L, are particularly interesting because they directly recognize a plethora of pathogens, including bacteria, viruses, and parasites. Both genes, which result from an ancient duplication, possess a neck region, made up of seven repeats of 23 amino acids each, known to play a major role in the pathogen-binding properties of these proteins. To explore the extent to which pathogens have exerted selective pressures on these innate immunity genes, we resequenced them in a group of samples from sub-Saharan Africa, Europe, and East Asia. Moreover, variation in the number of repeats of the neck region was defined in the entire Human Genome Diversity Panel for both genes. Our results, which are based on diversity levels, neutrality tests, population genetic distances, and neck-region length variation, provide genetic evidence that CD209 has been under a strong selective constraint that prevents accumulation of any amino acid changes, whereas CD209L variability has most likely been shaped by the action of balancing selection in non-African populations. In addition, our data point to the neck region as the functional target of such selective pressures: CD209 presents a constant size in the neck region populationwide, whereas CD209L presents an excess of length variation, particularly in non-African populations. An additional interesting observation came from the coalescent-based CD209 gene tree, whose binary topology and time depth (approximately 2.8 million years ago) are compatible with an ancestral population structure in Africa. Altogether, our study has revealed that even a short segment of the human genome can uncover an extraordinarily complex evolutionary history, including different pathogen pressures on host genes as well as traces of admixture among archaic hominid populations.     

Codon usage bias: causative factors,quantification methods and genome‐wide patterns: with emphasis on insect genomes

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole‐genome sequencing of numerous species, both prokaryotes and eukaryotes, genome‐wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole‐genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome‐sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome‐sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance.     

Genome scans reveal candidate regions involved in the adaptation to host plant in the pea aphid complex

A major goal in evolutionary biology is to uncover the genetic basis of adaptation. Divergent selection exerted on ecological traits may result in adaptive population differentiation and reproductive isolation and affect differentially the level of genetic divergence along the genome. Genome‐wide scan of large sets of individuals from multiple populations is a powerful approach to identify loci or genomic regions under ecologically divergent selection. Here, we focused on the pea aphid, a species complex of divergent host races, to explore the organization of the genomic divergence associated with host plant adaptation and ecological speciation. We analysed 390 microsatellite markers located at variable distances from predicted genes in replicate samples of sympatric populations of the pea aphid collected on alfalfa, red clover and pea, which correspond to three common host‐adapted races reported in this species complex. Using a method that accounts for the hierarchical structure of our data set, we found a set of 11 outlier loci that show higher genetic differentiation between host races than expected under the null hypothesis of neutral evolution. Two of the outliers are close to olfactory receptor genes and three other nearby genes encoding salivary proteins. The remaining outliers are located in regions with genes of unknown functions, or which functions are unlikely to be involved in interactions with the host plant. This study reveals genetic signatures of divergent selection across the genome and provides an inventory of candidate genes responsible for plant specialization in the pea aphid, thereby setting the stage for future functional studies.     

Genetic divergence with ongoing gene flow is maintained by the use of different hosts in phytophagous ladybird beetles genus Henosepilachna

Adaptation to different environments can promote population divergence via natural selection even in the presence of gene flow – a phenomenon that typically occurs during ecological speciation. To elucidate how natural selection promotes and maintains population divergence during speciation, we investigated the population genetic structure, degree of gene flow and heterogeneous genomic divergence in three closely related Japanese phytophagous ladybird beetles: Henosepilachna pustulosa, H. niponica and H. yasutomii. These species act as a generalist, a wild thistle (Cirsium spp.) specialist and a blue cohosh (Caulophyllum robustum) specialist, respectively, and their ranges differ accordingly. The two specialist species widely co‐occur but are reproductively isolated solely due to their high specialization to a particular host plant. Genomewide amplified fragment‐length polymorphism (AFLP) markers and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences demonstrated obvious genomewide divergence associated with both geographic distance and ecological divergence. However, a hybridization assessment for both AFLP loci and the mitochondrial sequences revealed a certain degree of unidirectional gene flow between the two sympatric specialist species. Principal coordinates analysis (PCoA) based on all of the variable AFLP loci demonstrated that there are genetic similarities between populations from adjacent localities irrespective of the species (i.e. host range). However, a further comparative genome scan identified a few fractions of loci representing approximately 1% of all loci as different host‐associated outliers. These results suggest that these three species had a complex origin, which could be obscured by current gene flow, and that ecological divergence can be maintained with only a small fraction of the genome is related to different host use even when there is a certain degree of gene flow between sympatric species pairs.     

The nuclear transcription factor PKNOX2 is a candidate gene for substance dependence in European-origin women

Substance dependence or addiction is a complex environmental and genetic disorder that results in serious health and socio-economic consequences. Multiple substance dependence categories together, rather than any one individual addiction outcome, may explain the genetic variability of such disorder. In our study, we defined a composite substance dependence phenotype derived from six individual diagnoses: addiction to nicotine, alcohol, marijuana, cocaine, opiates or other drugs as a whole. Using data from several genomewide case-control studies, we identified a strong (Odds ratio  = 1.77) and significant (p-value = 7E-8) association signal with a novel gene, PBX/knotted 1 homeobox 2 (PKNOX2), on chromosome 11 with the composite phenotype in European-origin women. The association signal is not as significant when individual outcomes for addiction are considered, or in males or African-origin population. Our findings underscore the importance of considering multiple addiction types and the importance of considering population and gender stratification when analyzing data with heterogeneous population.